Recombinant materials for production of a protein toxic to fire ants

ABSTRACT

A nucleic acid derived from  Bacillus thuringiensis  contains a nucleotide sequence that encodes a polypeptide demonstrated to be toxic to fire ants.

CROSS-REFERENCES TO RELATED APPLICATIONS

This Application for Patent claims the benefit of priority from, andhereby incorporates by reference the entire disclosure of, co-pendingU.S. Provisional Application for Patent Ser. No. 60/161,495, filed Oct.26, 1999.

TECHNICAL FIELD OF THE INVENTION

This invention relates to methods and compositions for controllingpopulations of Hymenopteran insect pests in the Formicidae (ant) familyusing a novel Bacillus thuringiensis (“PT”) toxin and preparation. Inparticular, the invention relates to effective methods of controllingpopulations of various fire ants of the family Solenopsis using a BTtoxin effective in killing fire ants, and a novel strain of BT producingsuch toxin.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with uses of Bacillus thuringiensis toxins as fire antbiocidal agents as an example. The imported fire ant, Solenopsisinvicta, is an introduced species that arrived in Mobile, Ala. fromSouth America around the 1930s. The imported fire ant has spread rapidlyacross the southern United States and continues to expand into areas ofNorth America with mild climates and adequate moisture and food.

The imported fire ant is an agriculturally and medically important pestharmful to domestic animals, wildlife and humans. Fire ants particularlythreaten obligate ground dwelling species and young animals of allspecies. The imported fire ant is thought to be responsible for thedecline of several native species. Proliferation of the imported fireant has been largely unchecked due to the absence of predators,pathogens and parasites that control its numbers in its nativeenvironment. The fire ant typically causes painful stings in humans andmore severe reactions may occur in allergic individuals. High densitiesof fire ants have been responsible for damage to roads, pastures, andelectrical and mechanical equipment. The recent appearance of multiplequeen colonies has made control of fire ant populations even moredifficult. An effective compound and method for control of fire antpopulations that is safe to users and consumers is urgently needed.

Past attempts at fire ant control have involved the highly toxicchlorinated hydrocarbons heptachlor, dieldrin, and Mirex. The EPA hasoutlawed the use of these highly toxic albeit relatively effectivecompounds for all but exceptional applications. The most commonly usedmodern control methods used include the chemicals hydramethylnon,avermectin, and the synthetic insect growth regulator fenoxycarb. Thesecompounds must be regularly reapplied and have not been able tosignificantly impact severe fire ant infestations or to control furtherspread. The search for control methods is now being conducted in theareas of sterile insect release and the introduction of natural enemies,both of which are only potential solutions with uncertain outcomes.

Bacillus thuringiensis (“BT”) is the genus and species of a large numberof strains of gram-positive, spore-forming bacteria which, under certainconditions, form a parasporal crystal comprised of insecticidal proteintoxin (Bulla, et al., Crit. Rev. Microbiol., 8:147-204, (1980); Höfteand Whitely, Microbiol Rev (1989) 53:242, (1989). The toxin itself is aglycoprotein product of cry genes (“Cry” is used to denote the protein;“cry” is used to denote the gene) as described by Höfte (Id.). Becausethe effects of the various Cry proteins are mediated by binding tounique receptors, the species specificity of a given BT toxin istypically quite limited as exemplified by the original classificationproposed by Höfte (Id.): CryI (Lepidopteran specific); CryII(Lepidopteran and Dipteran specific); CryIII (Coleoptera specific); andCryIV (Diptera specific). The BT toxins function in the brush border ofinsect midgut epithelial cells and, although highly insecticidal tocertain insects, are non-toxic to other organisms lacking toxin bindingreceptors (Gill, S. S. et al. Ann. Rev. Entomol (1992) 37:615).

Keen interest in BT toxins over the last 30 years has resulted in theisolation of more than 100 different BT crystal protein genes and thedevelopment of bioinsecticides for the control of insect species in theorders Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera,Orthoptera and Mallophaga and against nematodes, mites and protozoa(Schnepf et al., Microbiol. Mol. Biol. Rev. (1998) 62(3):775). BT toxinin various forms now accounts for 90% of the world sales of non-chemicalinsecticides.

U.S. Pat. Nos. 5,260,058, 5,268,297, 5,596,071 and 5,824,792, discloseprocesses and compositions for controlling pharoah ants (Monomoriumpharaonis) using toxin containing bacterial cells of various BT strains.Although these toxins are alleged to be effective against allHymenoptera and all ants, no testing beyond the pharoah ant wasperformed. Further, the protoxins of this invention were much larger(120-140 kD) than that described herein.

SUMMARY OF THE INVENTION

Prior to this invention, no BT toxins have been reported that are knownto be effective in controlling fire ants, nor are any BT toxinscommercially available for this indication. What is needed is a biocidalcomposition that is effective in controlling populations of the fireant, yet is not toxic to non-insect organisms. Such a biocide would havebroad applicability including the agricultural, domestic, environmentaland biomedical arenas. For this reason a BT toxin effective in killingfire ants would be particularly desirable.

Fire ants are omnivorous, although a large portion of their dietcomprises invertebrates which the fire ants sting and kill. They alsofeed on dead animal and plant tissues, seeds, developing and ripefruits, and are attracted to honeydew and sap flows. They are attractedto sugars, certain amino acids, ions in solution, and to some oilscontaining polyunsaturated fatty acids in these food sources.

Worker ants can only consume liquid foods, and nearly half of theresources that are returned to the nest are in the form of liquids.Liquids consumed and stored by the foraging workers are fed to otherworkers through trophallaxis. Once the worker arrives back at thecolony, the oils are slowly transferred to nurses and from the nurses tolarvae. Soluble sugars in addition to some soluble protein and aminoacid mixtures are strongly attractive and encourage trophallaxis amongworkers. This both dilutes the solution and reduces the speed ofmovement of these nutrients to larvae.

Undissolved solids greater than 0.88 microns are screened from theliquid in the pharynx of the worker fire ant and cannot be ingested bythe worker ants. The solids accumulate in the buccal region as pelletsand are later expelled to feed fourth-instar stage larvae, which areable to consume particles as large as 45 microns. Because solid food maybe used by the mature larvae but not the workers, solids move from thefield to these larvae more quickly and directly than liquid foods. Afterprocessing by the fourth instar larvae, previously solid foods can beutilized by the queen and young larvae through trophallaxis. As it isdesirable that worker ants transport poisons back to the colony anddistribute the poison throughout the colony, consideration to thefavored foods of fire ants together with the physical size of the poisonmay influence the efficacy of treatment modalities.

The present invention provides a BT protein that is active againstmembers of the Formicidae family, in particular the imported fire ant,Solenopsis invicta, and related species. Generally, the inventionprovides BT preparations that are effective in reducing populations offire ants and related species. The invention further provides novel B.thuringiensis strains that produce toxins effective against fire antsand related species. Effectiveness is defined as the ability to reducethe numbers of ants within a local fire ant population, typically amound-type community, by killing immature and/or mature individualswithin the population who are exposed to the toxin. Exposure may takethe form of ingestion of the subject toxin either directly in a baitformulation or as expressed in a food source or by trophallaxis.

This invention provides biologically pure cultures of an isolated BTbacterial cell having the identifying characteristics of strain UTD-001(NRRL No. B-30356).

In an alternate embodiment, the invention further provides recombinantDNA derived from the BT strain having the identifying characteristics ofUTD-001 and encoding BT Cry toxin proteins. These characteristicsinclude an approximately 73 kD wild type Cry protoxin protein that isactivated on proteolytic processing to an approximately 67 kD toxin, hastoxic activity against fire ants, has activity between pH 5-7, anSDS-PAGE profile substantially as shown in FIG. 2, and anarbitrary-primed PCR product profile substantially as shown in FIG. 3.

The DNA encoding BT Cry toxins may be isolated and cloned as recombinantDNA by the methods known in the art such as for example in the standardcloning manual of Sambrook, et al., Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 2nd ed.1989). The cry gene from UTD-001 has been cloned, and the sequences ofthe invention are identified as follows:

SEQ ID NO: Nucleic acid sequence for the cry 1 protoxin gene cloned fromUTD-001. SEQ ID NO: Amino acid sequence encoded by SEQ ID 2 NO: 1including active protein from residues 56 or 58 to 644. SEQ ID NO:Forward primer cry3A-F used to clone SEQ 3 ID NO: 1;ATGAATCCGAACAATCGAAG SEQ ID NO: Reverse primer cry3A-R used to clone SEQ4 ID NO: 1; TTAATTCACTGGAATAAATTC.

The invention further provides vectors for the cloning and expression ofrecombinant DNA encoding BT Cry protoxins or toxins that are toxic tofire ants. According to methods known in the art, the vectors may beadapted for expression of the Cry protoxin or toxin protein in varioushost organisms as desired. The invention contemplates the use of theseexpression vectors to produce novel host organisms which are engineeredto express the encoded proteins either transiently, upon induction, orconstitutively. Host organisms include those that are merely used asfactories for the production of large quantities of toxin which is thenpurified and formulated into a bait. Alternatively, host organismsinclude organisms engineered to both express the protein and to serve asa food source for fire ants. Host organisms may include prokaryoticcells and eukaryotic cells and organisms such as for example, yeast,fungi, plants or animals.

The invention further provides purified Cry protoxins and toxins andcompositions of these proteins that are active against Hymenoptera,Formicidae, and especially Solenopsis. In one embodiment, the Cryprotoxin disclosed herein is characterized by a molecular weight ofabout 73 kD by SDS-PAGE, and 72.9 kD by calculation from theconceptually translated protein. The protoxin is putatively cleaved atresidue 55 (between EA) by papain, and at residue 57 (between LD) bytrypsin to produce an active protein. Thus, the protoxin is reduced to atoxin of about 67 kD by proteolytic digestion. However, it is expectedthat this cleavage site is somewhat flexible resulting in smallvariations in size of the active toxin. The novel Cry toxin of thepresent invention may also be described as a Cry3A-like toxin based onits close homology to the Cry3A toxin of Btt.

In one such embodiment, the Cry toxin is toxic to Formicidae, includingSolenopsis invicta, S. richteri, S. xyloni, S. geminatam, S. geminata(aka Atta geminata Fabricius or S. geminata Mayr, S. japonica (aka S.fugax var. japonica), S. saevissima, S. orbuloides, S. punctaticeps, avariety of Solenopsis species identified by number, and related fire antspecies. The invention provides methods for reducing ant populations bycompositions containing Cry toxin or protoxin produced by BT strainshaving the identifying characteristics of UTD-001.

In yet another embodiment of the present invention, a biocide isproduced by (a) propagating B. thuringiensis microorganisms having theidentifying characteristics of strains UTD-001 under conditions whereinCry protoxin is produced; (b) purifying the Cry protoxin; and (c)formulating the purified Cry protoxin into a biocide effective againstants. The Cry protoxin may be pre-activated by proteolytic digestionprior to formulating it into a bait, or may be produced directly intoxin form by the host organism.

In a further embodiment, the invention provides methods for isolatingand cloning the BT toxin receptor of the imported fire ant and for usingsuch receptor to engineer toxins with greater potency against the fireant and for avoiding any resistance which may develop subsequent to theapplication of the present invention to fire ant populations.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete understanding of the method and apparatus of the presentinvention may be obtained by reference to the following DetailedDescription when taken in conjunction with the accompanying Drawingswherein:

FIG. 1 is a Coomassie blue stained SDS-PAGE gel of the cytoplasmicproteins isolated from UTD-001 and a control species, B. thuringiensis,subs. tenebrionis (Btt). The left panel is proteins from vegetativecells, whereas the right panel is from sporulating cells. The patternsclearly demonstrate that the UTD-001 strain has a Cry toxin ofapproximately the same size (about 67 kD) as the Cry3A protein of Btt.Molecular weight size markers (kD) are also indicated;

FIG. 2 is an ethidium bromide stained agarose gel of arbitrarily-primedPCR products of UTD-001 and the control species Btt. The arbitraryprimer was ACGCGCCCT. The arrows indicate bands present in the Bttstrain, that are absent in the UTD-001 strain. Size markers M (100 bpladder) are indicated on the right;

FIG. 3 shows a fire ant toxin assay where purified papain treated toxinis compared against purified toxin, crude spores & crystal toxin, andcontrols;

FIGS. 4A and 4B are the SDS-PAGE profiles of the activated toxin (4A)and crude spore and crystal toxin (4B) The 73 kD protoxin and 67 kDtoxin are both seen in 4B. The molecular weight markers (kD) are alsoindicated;

FIG. 5 illustrates the cloning of the Cry toxin gene from a 1.8 kb PCRproduct of UTD-001 into the TA cloning vector pCR2.1 (INVITROGEN™) toproduce the pCCRY3 plasmid. Primers used were Forward:ATGAATCCGAACAATCGAAG (SEQ. ID. NO: 3) and Reverse: TTAATTCACTGGAATAAATTC(SEQ. ID. NO: 4);

FIG. 6 is the nucleotide sequence (SEQ ID NO: 1) of the Cry protoxin ofUTD-001; and

FIG. 7 is the amino acid sequence (SEQ ID NO: 2) of the Cry protoxin ofUTD-001. The active toxin portion is in bold face. The arrow indicatesputative cleavage sites for the protoxin.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EXEMPLARY EMBODIMENTS

The present invention will now be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. This invention may, however, be embodied inmany different forms and should not be construed as limited to theembodiments set forth herein; rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

Abbreviations and Definitions

The following abbreviations and definitions are used throughout thisapplication: BT—Bacillus thuringiensis (B. thuringiensis); Btt—Bacillusthuringiensis subsp. tenebrionis; Cry toxin—toxin derived fromparasporal crystalline protein; cry—the gene encoding Cry toxin;EDTA—disodium ethylenediaminetetraacetate, dehydrate; kD—kilodaltons;LB—Luria broth; LC₅₀—lethal concentration resulting in a 50% mortality;PMSF—phenylmethane sulfonyl fluoride; and SDS-PAGE—sodium dodecylsulfate polyacryamide gel electrophoresis.

The term “x% homology” refers to the extent to which two nucleic acid orprotein sequences are identical as determined by BLAST homologyalignment as described by T. A. Tatusova & T. L. Madden (1999), “Blast 2sequences—a new tool for comparing protein and nucleotide sequences,”FEMS Microbiol Lett. 174:247-250 and using the following parameters:Program (blastn) or (blastp) as appropriate; matrix (OBLOSUM62), rewardfor match (1); penalty for mismatch (−2); open gap (5) and extension gap(2) penalties; gap x- drop off (50); Expect (10); word size (11); filter(off). An example of a web based two sequence alignment program usingthese parameters is found at www.ncbi.nlm.nih.gov/gorf/bl2.html. Highstringency is defined as including a final wash of 0.2×SSC at atemperature of 60° C.

The present invention concerns a novel BT isolate and genes therefromthat encode a novel Hymenoptera-active protein. In particular, thetoxins are active against the Formicidae and especially the Solenopsis.The novel BT isolate, known herein as Bacillus thuringiensis UTD-001 aswell as protoxins and toxins from this isolate, may be used to controlpests such as fire ants, carpenter ants, Argentine ants, and pharaohants.

The BT toxin gene from isolate UTD-001 has been cloned and sequenced andis presented in FIG. 6 (SEQ ID Nos.: 1 and 2). The coding region is fromnucleotides 1 to end (1936). The protein in FIG. 7 is 644 amino acids,and includes an pro-region from amino acids 1 to 55 or 57 and an activeregion from amino acids 56 or 58 to the end. The pI of the protoxin is5.67. The cry gene is 98% homologous to the cry3A gene of Btt, whichappears to be its closest relative at this time.

The novel toxin disclosed herein is active against ants, especially fireants. The present invention provides those of skill in the art with aSolenopsis-active toxin, methods for using the toxin, and gene thatencodes the toxin. The gene or gene fragment that encodes theSolenopsis-active toxin may be cloned and the gene fragment encoding thetoxin gene may be transferred to suitable hosts via a recombinant DNAvector.

Because the Cry toxins generally have a very broad range of speciesspecificity, it is anticipated that the Cry toxins of the invention willhave activity against other Hymenoptera. Species specificity studieswill be performed to confirm this prediction.

Recombinant hosts for use as ant food sources comprising the Cry toxinsof the invention may include a wide variety of microbial or plant hosts.Expression of the toxin gene results, directly or indirectly, in theproduction of the pesticide toxin. An important feature of thisembodiment of the invention is that the toxin produced should not form acrystal within the host, as is the norm in BT, because fire ants filterout large particles, such as the crystal, while eating. Therefore, thehost must not produce a crystal that is larger that the particle sizethat fire ants exclude during the eating process (e.g. 0.88 microns). Ofcourse, the cry gene may be engineered to produce the active, soluble 67kD form of the toxin which circumvents the size exclusion problem.

With suitable microbial hosts, e.g., Pseudomonas, the microbes may beapplied to the situs of the pest, where they will proliferate and beingested. The result is effective control of the fire ant. Factors thatmay be considered in selecting a host cell for Bacillus thuringiensistoxin production according to the present invention include: gene-hostcompatibility, ease of host transformation; availability of expressionsystems; efficiency of expression; stability of the toxin in the host;the presence of auxiliary genetic capabilities and whether the host isto function as a direct food source for the target insect, or merelyfunction as a source of toxin. Other factors that may be consideredinclude: pathogenicity of the host to animals and humans; ease offormulation and handling, economics, storage stability, and the like.Microorganism food source hosts are selected that are known to occupythe “phytosphere” (phylloplane, phyllosphere, rhizosphere, and/orrhizoplane) of one or more crops of interest. Generally, the organismshould provide stable maintenance and expression of the gene and providefor improved protection of the toxin from environmental degradation andinactivation.

A large number of microorganisms are known to inhabit the phylloplane(the surface of the plant leaves) and/or the rhizosphere (the soilsurrounding plant roots) of a wide variety of important crops and are aknown food source for the fire ant. These microorganisms includebacteria, algae, and fungi. Of particular interest are microorganisms,such as bacteria, e.g., genera Pseudomenas, Erwinia, Serratia,Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas,Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter,Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast,e.g., genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces,Rhodotorula, and Aureobasidium.

Other organisms that may be used as a food source by fire ants includephytosphere bacterial species, such as: Pseudomonas syringae, P.fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacteriumtumefaciens, Rhodopseudomonas spheroides, Xanthomonas campestris,Rhizobium melioti, Alcaligenes entrophus, and Azotobacter vinlandii; andphytosphere yeast species such as Rhodotorula rubra, R. glutinis, R.marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii,Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomycesroseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans.Of particular interest are the pigmented microorganisms.

The BT strains of the present invention may be cultured using standardart media and fermentation techniques, however, certain specificconditions are disclosed herein that fall within the standard art. Uponcompletion of a fermentation cycle, the bacteria may be harvested byfirst separating the BT spores and parasporal crystals from thefermentation broth by means well known in the art. The recovered BTspores and crystals may be formulated into a wettable powder, liquidconcentrate, granules, or other formulations by the addition ofsurfactants, dispersants, inert carriers and other components tofacilitate handling and application for particular target pests.

Not generally known in the art, and specifically recognized herein, isthe fact that fire ants specifically filter large objects such asbacterial spores and crystals away from the oral cavity and prevententry of these large items into the digestive system. Therefore, it isimportant for fire ant solid bait preparations that particle size belimited to 0.88 microns or less, to allow the fire ant to effectivelyingest the poison, but particle size is less relevant for otherHymenopteran insects. Alternatively, the BT spores and protoxin crystalscan be at least partially purified and partially dissolved in a liquid,or the crystal size reduced by grinding or genetic modification, or thetoxin expressed as soluble, active proteins to circumvent the sizeexclusion mechanism of fire ant defense.

Upon complete or partial purification, the pesticide toxin of thepresent invention may be provided in a concentration for the amount ofliquid or dissolving liquid needed. The concentration of toxin will varywidely depending upon the nature of the particular formulation. Thepesticide may be made available to fire ants at a concentration of about1% weight to volume, however, it may be at 100% by weight if provided ina dry form with an attractant. The attractant may even be the toxinitself or a blotting agent or powder on which the toxin is provided. Dryformulations, for example, will have from about 1-95% by weight of thepesticide whereas the liquid formulations will generally be from about1-60% by weight of the solids in the liquid phase. The formulations maybe applied to the environment of the Hymenopteran pest(s), e.g., plants,soil or water, by spraying, dusting, sprinkling, baits or the like.

EXAMPLE 1 Isolation of Bacillus Thuringiensis Strains

A 1 gram sample of soil was mixed with 10 ml of LB buffered with 0.25 Msodium acetate, pH 6.8 and incubated with strong agitation (^(˜)200 rpm)overnight at 30° C. A 1.5 ml aliquot of the sample was removed and heatshocked at 80° C. with agitation in a 50 ml conical tube for 3 minutes.Aliquots of 10, 20 and 100 μl were then removed and plated on LB mediumovernight at 30° C. The following day colonies were picked and platedonto GYS plates and incubated at 30° C. overnight. The GYS medium isprepared as follows:

MODIFIED “GYS” MEDIUM Per Liter Glucose  2.0 g (NH₄)₂SO₄  2.0 g CaCl₂(Anhydrous) 0.08 g MgSO₄ (Anhydrous) 0.20 g MnSO₄ (Monohydrate) 0.05 gCitric Acid  1.5 g Yeast Extract  2.0 g pH may be adjusted with KOH to7.5 and autoclaved after the desired pH is attained.

Isolates were examined microscopically to determine if crystallineinclusions were produced. Inclusions were visualized by staining withmalachite green/Gram Safranin as follows: cells were dried onto a slideand stained for 15 minutes with a 5% aqueous solution of malachite greenover boiling H₂O. The slide was rinsed and stained for 5-6 minutes withGram Safranin Solution (stock solution is 2.5% solution of Safranin 0(SIGMA™) in 100 ml ETOH; the working solution is a 1:10 dilution ofstock solution in H₂O). The slide is rinsed and dried. Spores staingreen/blue while crystals and vegetative cells stain red.

Isolates which appeared to produce crystals were noted, grown in LBovernight at 30° C. and restreaked on GYS plates to obtain axeniccultures. Cultures were inspected again by staining with Gram Safraninto identify isolates producing crystals and spores. Such cultures weregrown 5 days in 7 ml of liquid GYS, stained again for crystal/sporeproduction and centrifuged and washed 3 times w/50 ml TrisCl, pH 7.5, 10mM KC1, and 10 mM EDTA. The pellets were lyophilized and weighed forlater use.

EXAMPLE 2 Cry Toxin Preparation

To prepare toxin, one ml of culture is centrifuged in a bench-topmicrofuge for one minute, resuspended in 200 μl of proteinsolubilization buffer and boiled for 10 minutes. The prepared sample isloaded on a 12% SDS-PAGE gel and electrophoresed. The total proteinprofile for each sample is determined by staining of the gel withCoomassie blue. Both vegetative and sporulating cells are analyzed inthis way, and an example is shown in FIG. 1. It is noted that because noprotease inhibitors were used to prepare the total protein extracts, theprotoxin has been entirely cleaved to the 67 kD form. In preparationswhere cleavage is incomplete, doublets may be seen, and in preparationswhere protease activity is completely prevented, a single 73 kD band isseen.

Isolates containing Cry toxin protein profiles of interest as determinedby SDS-PAGE are selected for bioassay. Isolates are grown in 1-literbatch cultures containing GYS broth. After complete sporulation of theculture, determined by malachite green staining, the cultures arecentrifuged and the parasporal crystals are washed using several roundsof sonification and buffer changes. The washed crystals are purifiedfurther by centrifugation on a sucrose gradient of 30-80%. The purifiedcrystals are solubilized in a sodium carbonate buffer and the protoxinconverted to activated toxin using papain or trypsin. The activatedtoxin is purified by HPLC or FPLC.

The purified active toxin is used in insect bioassay by adding varyingconcentrations to the insect diet as described above. In this way, astrain of fire ant effective BT called UTD-001 was identified. StrainUTD-001 was characterized according to its total protein bandingpatterns, as described above. The protein banding pattern was similar,but not identical, to the protein banding pattern of a controlspecies—Btt as shown in FIG. 1.

Btt is known to be effective against certain beetles, and contains aCry3A toxin. The Cry3A toxin is expressed as an approximately 73-kDprotoxin, which is activated by proteolytic cleavage to a 67 kD toxin.It should be understood to one of ordinary skill in the art that the Btttoxin exhibits toxicity properties, as illustrated in FIG. 3, forexample, Cry3A-like toxins. Further characterization of the UTD strainwas performed by arbitrary primer PCR (FIG. 2), a powerful method toidentify BT serivars and strains, Brousseau, C., et al., (1993) Appl.Environ. Microbiol. 59: 114-119. Again, the profiles are similar but notidentical to the control strain Btt. In particular, three bands(indicated by arrows) are present in the Btt strain, that are notpresent in the UTD-001 strain.

EXAMPLE 3 Purification and Activation of Cry Protoxin Crystals

A one-liter culture of a fire ant effective Btt strain is grown inmodified GYS medium for 4-6 days until most of the cells have lysed. Themedium containing the lysed cells is centrifuged for 20 minutes at 7500rpm. The supernatant is discarded and the cell pellet transferred to 40ml centrifuge tubes. The cell pellet is resuspended in 1M NaCl (30ml/tube) and centrifuged for 20 minutes at 15,000 rpm. Two subsequentwashes are done with distilled water. The cells (2-4 grams) areresuspended in two tubes in 68% RENOGRAFIN®-76 (20.4 ml Renografin®-76plus a solution of 3 ml 100 mM EDTA and 6.6 ml 2% Triton X-100 in dH₂O)(RENOGRAFIN®-76 is diatrizoate meglumine and diatrizoate sodiummanufactured by SQUIBB DIAGNOSTICS™, New Brunswick, N.J.).

The cells' debris is pelleted, for example, by centrifugation in a JA-20or Sorvall SS-34 rotor for 2 hours at 15,000 rpm. The supernatantcontaining the crystals is pipetted off and placed in a separatecentrifuge tube. The supernatant is filtered using 1.2-pm filter paperand, after addition of EDTA to 3-5 mM and PMSF 0.1 mg/ml, is dialyzedagainst distilled water for 36 hours using a dialysis membrane with amolecular weight cutoff of 12,000 to 14,000. The dialyzed material isthen centrifuged for 15 min at 7,500 rpm and the supernatant containingthe crystals is stored wet at 4° C.

The protoxin crystals are solubilized in 3.3 M NaBr, pH 7.0, and 50 mMphosphate buffer containing 1 mM PMSF. Insoluble papain beads (SIGMA™)are activated according to the manufacturer's instructions and added tothe solubilized crystal toxin to effect cleavage. Cleavage is arrestedby addition of Na-p-tosly-L-lysine chloromethyl ketone (TLCK) to 1.25mg/ml and 0.2 volume of pH 10.0 Na₂CO₃. Cleaved toxin is purified bypassage over Sephadex G-75 (AMERSHAM PHARMACIA BIOTECH, INC.™,Piscataway, N.J.) equilibrated with 50 mM Na₂CO₃, pH 10.0, containing 1mM EDTA.

EXAMPLE 4 Alternative Method of Purification and Activation of BT Toxin

Bacillus thuringiensis toxin can be isolated from recombinant cellsexpressing Cry toxin. For example, E. coli containing the cry gene isprepared. A 500 ml culture is then grown to an O.D.₆₀₀ of ^(˜)0.8. Wherethe recombinant gene is cloned under the influence of the lac promoter,expression is induced by treatment with 0.1 mMisopropyl-β-D-thiogalactopyranoside (IPTG) overnight at 37° C.

Cells are pelleted by centrifugation, for example at 5,000 rpm in aJA-14 rotor for 10 minutes. The cell pellet is resuspended in 25 ml ofTES buffer (50 mM Tris, pH 8.0; 50 mM EDTA; 15% sucrose). Cells arelysed by treatment with lysozyme (0.5 mg/ml final concentration) andPMSF (0.1 mM final concentration) for 30 minutes at room temperaturefollowed by sonication, 3×1 minute on ice. The supernatant and surfacedebris are discarded and the cells are suspended in 20 ml TTN buffer (20mM Tris, pH 8.0; 2% Triton X-100; 0.5 M NaCl). The pellets are firstsuspended with a spatula, then with a glass Dounce, and then pelleted at12,000 rpm for 15 minutes. This washing procedure is repeated for 4cycles. The pellet is then washed 2× with 20 ml PBS: Acetone (5:1) asabove followed by a wash in PBS (per liter: 8 gm NaCl; 0.2 gm KCl; 1.44gm Na₂HPO₄; 0.24 grm KH₂PO₄) alone. Finally, the pellet is resuspendedin 20 ml of Na₂CO₃ buffer (50 mM Na₂CO₃, pH 10.0; 5 mM DTT) and can bestored at 4° C. or is shaken at 37° C. for 4 hours to solubilize theprotoxin. The resulting milky solution is pelleted to remove insolublematerial, and additional sodium carbonate may be added if desired.

The protoxin preparation can be analyzed on a mini-gel and should show apredominantly 73-kD protoxin plus some smaller (bacterial) proteins.Protein can be quantified using the BCA protein reagent (PIERCE™) as perthe manufacturer's recommendations prior to papain treatment. Protoxinis stored at 4° C. Freezing results in deterioration of the protoxin.

Activation of the protoxin can alternatively be performed using trypsin.TPCK (SIGMA™) trypsin is added for a trypsin:protoxin ratio of 1:20 andthe mixture is shaken at room temperature for 15 minutes. The reactionmixture is placed on ice and an aliquot is checked for completedigestion on a mini-gel. If digestion is complete, PMSF is added to 100μM on ice.

The cleaved mixture is dialyzed overnight against 20 mM Tris with 100 mMNaCl, pH 9.5. Prior to final dialysis, concentration of activated toxinshould be no more than 2 mg/ml to avoid precipitation. The dialyzedpreparation is filtered through a 0.2 μm filter and subjected to furtherpurification by FPLC. The 18% B fraction on FPLC is collected using a100-1000 mM NaCl gradient as described below.

EXAMPLE 5 Purification of Cry Toxin by FPLC

FPLC procedures are conducted according to standard procedures includingthe following particulars.

Typical FPLC Profile (With HR5 Column at Port 3, with 1 ML Sample Loop):

0.0 CONC %B 0.0 (initial buffer is 100% A) 0.0 ML/MIN 1.0 (buffer flowrate) 0.0 CM/MIN 0.5 (chart speed as controlled by computer) 0.0PORT.SET 6.0 (sampler “off”) 0.0 VALVE.POS 1.1 (valve #1 bypass loop)0.0 VALVE.POS 2.3 (valve #2 leading to column at port 3) 0.0 VALVE.POS3.3 (valve #3 leading from column at port 3) 0.0 MIN/MARK 110 N/A 0.5VALVE.POS 1.2 (valve #1 open, loads loop onto column) 2.0 VALVE.POS 1.1(valve #1 closes after entire column has loaded on column, plus extra)7.0 CONC %B 0.0 (provides 5 minutes of Buffer A for flow- through) 37.CONC %B 30. (standard gradient of 1%/min) 40. CONC %B 100 (at 30% Bgradient then jumps to 100% in 3 minutes) 45. CONC %B 100 (5 minuteshold at 100% B) 45. CONC %B 0.0 (jump to 100% A) 60. CONC %B 0.0 (end ofmethod after 5 minute hold at 100% A) FPLC Buffer ‘A’ (low salt): 50 mMTris pH 10.0 100 mM NaCl 5% glycerol; filter through 0.2 μm filter. FPLCBuffer ‘B’ (high salt): 50 mM Tris pH 10.0 1-M NaCl 5% glycerol; filterthrough 0.2 μm filter.

EXAMPLE 6 Fire Ant Mortality

The invention described herein was targeted toward the identification ofBT effective in killing populations of the imported fire ant.Consideration was given to BT toxin characteristics together with thecharacteristics of the gut environment in which the toxin must exert itstoxic effect. Some BT strains produce toxins that work at neutral pH andlower (pH range of 5-7). The gut environments of different insects maybe characterized by different pH's. In particular, the gut of fire antsis slightly acidic. A BT toxin that is effective against fire ants mustsurvive in the fire ant gut environment.

FIG. 3 is a graph showing the mortality of the populations of theimported fire ant that were treated with various BT toxinconcentrations. Briefly, the procedure is as follows. A separate colonyof worker ants is used for testing each concentration. The variousconcentrations are combined in a 10% solution of sucrose in distilledwater for all of the tests. Prior to the test, the worker ant coloniesare held without food for 5 days. Then the 20 workers for eachrepetition are placed in 30-ml cups for 14 days. The toxin preparationsare placed in cups for only the initial 24 hours after that, they areremoved and replaced with a 10% sucrose in distilled water solution.Percent mortality is recorded at day 1, 2, 3, 6, 8, 10 and 14. Thecontrol is 10% sucrose in distilled water solution only.

LC₅₀ has not yet been conclusively determined, but preliminary resultsfrom 6 month old toxin of doubtful quality indicates that the LC₅₀ couldbe as low as 70 ng/ml. Studies are also planned to determine the speciesspecificity of the new Cry toxin. In particular, bees and wasps will beassayed for toxin efficacy.

EXAMPLE 7 Cloning of the Cry Gene from UTD-001

The cry gene of strain UTD-001 are contained on plasmids of similar sizeand restriction pattern as that shown by the control strain Btt (notshown). Hence, it is anticipated that the gene might be very similar tothe Btt gene. Thus, primers cry3A-F and cry3A-R (derived from the Bttcry gene) were used to amplify a 1.8-kb fragment of UTD-001 that has aTAQ created A overhang. The fragment is excised from a low-melt agarosegel and ligated into linearized pCR2.1 with a T overhang to produce thepCRRY3 plasmid (see FIG. 5—TA cloning method by INVITROGEN™).

The gene was sequenced and is shown in FIG. 6 along with theconceptually translated protoxin amino acid sequence in FIG. 7. Thecoding region extends from nucleotides 56 or 58 to the end. The putativeproteolytic cleavage site is indicated by the arrow and the toxinsequence is indicated in bold. The calculated molecular weight of theprotoxin is 72.9 kD, and the toxin is about 67 kD, which agrees wellwith the sizes as determined by SDS-PAGE.

The gene shows greatest homology (98%) to the Cry3A gene of the Bttstrain used as a control. In particular, the amino acid sequence (SEQ IDNO: 2) was Searched by BLASTP under the search parameters describedunder definitions above and homologies to a number of Coleopteraspecific, 73-74 Kd Cry protoxins were found. The closest homology was tothe Cry3A(A) toxin (Acc. No. P07130) from B. thuringiensis var.tenebrionis with homologies ranging from 632/644 (98%) to 572/584 (97%)amino acid identities from varying Btt isolates. The next closesthomologies were to the Cry3C(A) toxin of B. thuringiensis subsp.kurstaki (Acc. No. Q45744) at 479/652 (73%) amino acid identities, andthe Cry3B(B) toxin (Acc. No. Q06117) with 439/650 (67%) identities.Similarly, the nucleotide sequence showed 1902/1936 (98%) nucleotidehomology to the Cry3A(A) sequence of Btt.

Because the novel cry toxin was surprisingly closely related to Btttoxin, a toxin known to only be effective against Coleoptera, the Btttoxin was also tested for efficacy against fire ants. Preliminaryresults indicate that the Btt toxin is effective against the fire ant,although with less efficacy than the strain described herein. Thus, anew use for the prior Btt toxin has been unexpectedly discovered. Thisfire ant activity may have been previously obscured because of theunusual dietary requirements of the fire ant, which cannot consume largesolids.

All references cited herein are hereby incorporated by reference.Although this invention has been described in reference to illustrativeembodiments, this description is not intended to be construed in alimiting sense. Various modifications and combinations of theillustrative embodiments, as well as other embodiments of the invention,will be apparent to persons skilled in the art upon reference to thedescription. It is, therefore, intended that the appended claimsencompass any such modifications or embodiments.

4 1 1936 DNA Bacillus thuringiensis 1 atgaatccga acaatcgaag tgaacatgatacaataaaaa ctactgaaaa taatgaggtg 60 ccaactaacc atgttcaata tcctttagcggaaactccaa atccaacact agaagattta 120 aattataaag agtttttaag aatgactgcagataataata cggaagcact agatagctct 180 acaacaaaag atgtcattca aaaaggcatttccgtagtag gtgatctcct aggcgtagta 240 ggtttcccgt ttggtggagc gcttgtttcgttttatacaa actttttaaa tactatttgg 300 ccaagtgaag acccgtggaa ggcttttatggaacaagtag aagcattgat ggatcagaaa 360 atagctgatt atgcaaaaaa taaagctcttgcagagttac agggccttca aaataatgtc 420 gaagattatg tgagtgcatt gagttcatggcaaaaaaatc ctgtgagttc acgaaatcca 480 catagccagg ggcggataag agagctgttttctcaagcag aaagtcattt tcgtaattca 540 atgccttcgt ttgcaatttc tggatacgaggttctatttc taacaacata tgcacaagct 600 gccaacacac atttattttt actaaaagacgctcaaattt atggagaaga atggggatac 660 aaaaaagaag atattgctga atttttaaaaagacaactaa aacttaccca ggaatatact 720 gaccattttg ttcaatggta ttatgttggattagataaaa ttagaggttc attctatgaa 780 tcttgggtaa actttaaccg ttatcgcagagagatgacat taacagtatt agatttaatt 840 gcactatttc cattgtatga tgttcggctatacccaaaag aagttaaaac cgaattaaca 900 agagacgttt taacagatcc aattgtcggagtcaacaacc ttaggggcta tggaacaacc 960 ttctctaata tagaaaatta tattcgaaaaccacatctat ttgactatct gcatagaatt 1020 caatttcaca cgcggttcca accaggatattatggaaatg actctttcaa ttattggtcc 1080 ggtaattatg tttcaactag accaagcataggatcaaatg atataatcac atctccattc 1140 tatggaaata aatccagtga acctgtacaaaatttaggat ttaatggaga aaaagtctat 1200 agagccgtag caaatacaaa tcttgcggtctggccgtccg ctgtaaattc aggtgtaaca 1260 aaagtgaaat ttagccaata taatgatcaaacagatgaag caagtacaca aacgtcggac 1320 tcaaaaagaa atgttggcgc ggtcagctgggattctatcg atcaattgcc tccagaagca 1380 acagatgaac ctctagaaaa gggatatagccatcaactca attatgtaat gtgcttttta 1440 atgcagggta gtagaggaac aatcccagtgttaacttgga cacataaaag tgtagacttt 1500 tttaacatga ttgattcgaa aaaaattacacaacttccgt tagtaaaggc atataagtta 1560 caatctggtg cttccgttgt cgcaggtcctaggtttacag gaggagatat cattcaatgc 1620 acagaaaatg gaagtgcggc aactatttacgttacaccgg atgtgtcgta ctctcaaaaa 1680 tatcgagcta gaattcatta tgcttctacatctcagataa catttacact cagtttagac 1740 ggggcaccat ttaatcaata ctatttcgataaaacgataa ataaaggaga cacattaacg 1800 tataattcat ttaatttagc aagtttcagcacaccattcg aattatcagg gaataactta 1860 caaataggcg tcacaggatt aagtgctggagataaagttt atatagacaa aattgaattt 1920 attccagtga attaaa 1936 2 644 PRTBacillus thuringiensis 2 Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr IleLys Thr Thr Glu 1 5 10 15 Asn Asn Glu Val Pro Thr Asn His Val Gln TyrPro Leu Ala Glu Thr 20 25 30 Pro Asn Pro Thr Leu Glu Asp Leu Asn Tyr LysGlu Phe Leu Arg Met 35 40 45 Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp SerSer Thr Thr Lys Asp 50 55 60 Val Ile Gln Lys Gly Ile Ser Val Val Gly AspLeu Leu Gly Val Val 65 70 75 80 Gly Phe Pro Phe Gly Gly Ala Leu Val SerPhe Tyr Thr Asn Phe Leu 85 90 95 Asn Thr Ile Trp Pro Ser Glu Asp Pro TrpLys Ala Phe Met Glu Gln 100 105 110 Val Glu Ala Leu Met Asp Gln Lys IleAla Asp Tyr Ala Lys Asn Lys 115 120 125 Ala Leu Ala Glu Leu Gln Gly LeuGln Asn Asn Val Glu Asp Tyr Val 130 135 140 Ser Ala Leu Ser Ser Trp GlnLys Asn Pro Val Ser Ser Arg Asn Pro 145 150 155 160 His Ser Gln Gly ArgIle Arg Glu Leu Phe Ser Gln Ala Glu Ser His 165 170 175 Phe Arg Asn SerMet Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val Leu 180 185 190 Phe Leu ThrThr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu Leu 195 200 205 Lys AspAla Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Lys Lys Glu Asp 210 215 220 IleAla Glu Phe Leu Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr 225 230 235240 Asp His Phe Val Gln Trp Tyr Tyr Val Gly Leu Asp Lys Ile Arg Gly 245250 255 Ser Phe Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met260 265 270 Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr AspVal 275 280 285 Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg AspVal Leu 290 295 300 Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly TyrGly Thr Thr 305 310 315 320 Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys ProHis Leu Phe Asp Tyr 325 330 335 Leu His Arg Ile Gln Phe His Thr Arg PheGln Pro Gly Tyr Tyr Gly 340 345 350 Asn Asp Ser Phe Asn Tyr Trp Ser GlyAsn Tyr Val Ser Thr Arg Pro 355 360 365 Ser Ile Gly Ser Asn Asp Ile IleThr Ser Pro Phe Tyr Gly Asn Lys 370 375 380 Ser Ser Glu Pro Val Gln AsnLeu Gly Phe Asn Gly Glu Lys Val Tyr 385 390 395 400 Arg Ala Val Ala AsnThr Asn Leu Ala Val Trp Pro Ser Ala Val Asn 405 410 415 Ser Gly Val ThrLys Val Lys Phe Ser Gln Tyr Asn Asp Gln Thr Asp 420 425 430 Glu Ala SerThr Gln Thr Ser Asp Ser Lys Arg Asn Val Gly Ala Val 435 440 445 Ser TrpAsp Ser Ile Asp Gln Leu Pro Pro Glu Ala Thr Asp Glu Pro 450 455 460 LeuGlu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu 465 470 475480 Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His Lys 485490 495 Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln Leu500 505 510 Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val ValAla 515 520 525 Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr GluAsn Gly 530 535 540 Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser TyrSer Gln Lys 545 550 555 560 Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr SerGln Ile Thr Phe Thr 565 570 575 Leu Ser Leu Asp Gly Ala Pro Phe Asn GlnTyr Tyr Phe Asp Lys Thr 580 585 590 Ile Asn Lys Gly Asp Thr Leu Thr TyrAsn Ser Phe Asn Leu Ala Ser 595 600 605 Phe Ser Thr Pro Phe Glu Leu SerGly Asn Asn Leu Gln Ile Gly Val 610 615 620 Thr Gly Leu Ser Ala Gly AspLys Val Tyr Ile Asp Lys Ile Glu Phe 625 630 635 640 Ile Pro Val Asn 3 20DNA Bacillus thuringiensis 3 atgaatccga acaatcgaag 20 4 21 DNA Bacillusthuringiensis 4 ttaattcact ggaataaatt c 21

What is claimed is:
 1. An isolated or recombinant nucleic acid molecule,comprising a nucleotide sequence encoding a protein toxic to Formicidaewhich protein comprises residues 58 to 644 of SEQ ID NO:
 2. 2. Thenucleic acid molecule of claim 1, wherein the nucleotide sequenceencodes a precursor to a protein toxic to Formicidae which precursorcomprises SEQ ID NO:
 2. 3. The nucleic acid molecule of claim 2,comprising a nucleotide sequence of nucleotides 1 to 1936 of SEQ IDNO:
 1. 4. The nucleic acid molecule of claim 1, comprising a nucleotidesequence of at least nucleotides 174 to 1936 of SEQ ID NO:
 1. 5. Anucleic acid molecule which comprises an expression system forexpressing the nucleotide sequence of claim
 4. 6. Host cells modified tocontain the nucleic acid molecule of claim
 5. 7. A method to produce aprotein encoded by a nucleotide sequence comprising nucleotides 174-1936of SEQ. ID. NO: 1, which method comprises culturing the cells of claim 6so as to produce said protein.
 8. A nucleic acid molecule whichcomprises an expression system for expression of the nucleotide sequenceof claim 1 in a host cell.
 9. A host cell which contains the nucleicacid molecule of claim
 8. 10. A method to produce a protein comprisingresidues 58-644 of SEQ. ID. NO: 2, which method comprises culturing thecells of claim 9 so as to produce said protein.
 11. A nucleic acidmolecule, comprising a nucleotide sequence encoding a protein at least99% homologous over the entire length of residues 58 to 644 of SEQ IDNO: 2, wherein said protein is toxic to Formicidae.
 12. A nucleic acidmolecule which comprises an expression system for expressing thenucleotide sequence of claim
 11. 13. Host cells modified to contain thenucleic acid molecule of claim
 12. 14. A method to produce a protein atleast 99% homologous over the entire length of residues 58 to 644 of SEQID NO: 2, wherein said protein is toxic to Formicidae, which methodcomprises culturing the cells of claim 13 so as to produce said protein.